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Effect of peripheral clonidine on ingestive behavior
Physiology & Behavior, Vol. 21, pp. 73-77. Pergamon Press and Brain Research Publ., 1978. Printed in the U.S.A.
Effect of Peripheral Clonidine on Ingestive Behavior JEFFREY
ATKINSON,
E R N S T J. K I R C H E R T Z
AND LISE PETERS-HAEFELI
3
Institut de Pharmacologie de l'Universit~, 1011 Lausanne, Switzerland ( R e c e i v e d 14 D e c e m b e r 1977) ATKINSON, J., E. J. KIRCHERTZ AND L. PETERS-HAEFELI. Effect of peripheral clonidine on ingestive behavior. PHYSIOL. BEHAV. 21(1) 73--77, 1978.--The effect of parenteral elonidine, a central alpha-sympathomimetic agent, on eating and drinking was studied in rats. Small (37.5-75 /~g/kg) and large (150--300/~g/kg) doses of clonidine acutely depressed water intake for periods up to 6 hours. The antidispsogenic effect was followed by a delayed increase of fluid intake apparently due to the diuretic effect of the drug in rats, and this was suppressed by nephrectomy. With dally injections for periods up to 3 days large doses depressed both water and food intake. When dally injections were continued for more than 4 days, an appetite-stimulating effect was noted. It was concluded that clonidine primarily depresses water and food intake. The delayed, apparently dipsogenic, effect observed is due to a primary diuretic effect. The appetitestimulating effect of clonidine observed with chronic treatment may be a primary cerebral effect unmasked when tolerance develops to the (presumably) peripheral, anorectic effect. Clonidine
Food intake
Fluid intake
Diuresis
IT IS known that stimulation of central alpha-adrenergic receptors will provoke eating [12] and inhibit drinking [16]. Clonidine, an imidazoline derivative used as an antihypertensive drug, should share these properties, as: (a) it acts as an alpha-adrenergic agonist [5], and (b) has a marked central action [18]. Published reports on clonidine effects on food and fluid intake are, however, contradictory. It has been reported that clonidine, given intracerebrally, stimulates [3,13] or, given peripherally, inhibits food intake [11]; likewise for fluid intake, clonidine has been reported to have no effect when given intracerebrally [13] or to decrease intake when given peripherally [10]. When attempting to elucidate these contradictions, general points such as differences in administration route, doses used, etc. have to be taken into account, together with two specific points, peculiar to the effects of clonidine. The fu-st is that s.c. clonidine has a marked sedative effect [9] and thus, will induce a dose-dependent decrease in overall activity, possibly, including food intake. The second point is that clonidine has a marked, dosedependent diuretic effect, in the rat [5], and could, therefore, be expected, to induce various degrees of secondary drinking following a primary, dose-dependent, diuretic water loss. We have attempted to study the basic actions of clonidine on food and fluid intake and their possible perturbation by sedative and/or diuretic " s i d e " effects, in the following ways: 1. Using a wide range of clonidine doses from those possessing little sedative action to those inducing complete, long-lasting immobility. 2. Using rats incapable of losing water by diuresis (bilat-
eral nephrectomy or bilateral ureter ligation). 3. Following changes in water loss and water intake when clonidine is given chronically, as it has been reported that tolerance can develop to some of its effects e.g. its peripheral alpha-sympathomimetic [ 19] and central sedative [9] actions. METHOD
Animals Female Sprague-Dawley rats were maintained on a standard rat diet and tap water ad lib before each experiment.
Procedure Experiment 1: Short-term effects of parenteral clonidine on food andfluid intake. Rats of 250--260 g body weight were injected SC once daily (at 10:00 a.m.) with doses of 37.5, 75, 150 or 300/zg/kg clonidine for 3 days. They were allowed free access to a standard rat diet (pellets) and water. F o o d intake and body weight change over 3 days, and water intake during the 6 and 24 hr following each daily injection, were measured. Controls wre injected with the same volumes of solvent (0.9% NaC1, 5 ml/kg).
Experiment 2: Effect of lO days treatment with parenteral clonidine on food andfluid intake. Eight rats o f 188 --- 2 g initial body weight were housed in siliconized, polyethylene metabolic cages. Standard rat pellets and water were continuously available ad lib. and rats were injected daily (at 10:00 a.m.) with 0.9% NaCI (5 ml/kg) SC for a preliminary period of 18 days. F o o d and water intake and urine volume were measured 6 hr later, the rats were placed in clean cages,
tPreliminary communications see [1] and [7]. 2Research supported by Swiss National Science Foundation, Grants No. 3.053-074 and 3.028-0.76. 3Requests for reprints to J. Atkinson, Institut de Pharmacologie de l'Universit~, 1011 Lausanne, Switzerland.
C o p y r i g h t © 1978 B r a i n R e s e a r c h P u b l i c a t i o n s Inc.--0031-9384/78/0701-0073502.00/0
74
A T K I N S O N , K I R C H E R T Z AND P E T E R S - H A E F E L 1
WATER
PROBABILITY c d m[Ikg b.w (~)b 130(~ d b b INTAKE
120. ll0100-
~ ] HOURS 7 TO
4
HOURS 1 TO 6 AFTER CLONIDINE S.C.
90. 80.
60. 50.
40. 30. 20.
0
Experiment 4: Short-term effects of clonidine on food intake of hungry rats. Acute anorectic effects of drugs are magnified in animals given access to food after period of starvation. Experiments were, therefore, carried out in rats routinely starved for 18/24 hr. Rats of 190 to 200 g body weight were trained to eat a standard rat diet 6 hr per day (10:00 to 16:00) with tap water for 24/24 hr, ad lib. After 3 days, they stablized their food intake at 53 --+ 2 g/kg/6 hr (water intake was 103 _ _8 g/kg/24 hr). Food intake was, thus, approximately 75% of the unrestricted 24 hr intake. On the 4th day, they were distributed at random into 3 groups (of 6 rats each) and the groups were injected with 75 or 300 /xg/kg clonidine or 0.9% NaC1 SC. Free access to food and water was allowed from the time of the injection onwards, and food intake was measured 6 hr later.
70.
10
three groups and were subjected to one of the following operations (under ether anesthesia): bilateral nephrectomy: both kidneys removed via a single dorsal incision; ligation of ureters: both ureters tied off via a single ventral incision; sham: ventral incision, separation of ureters and kidneys from other viscera. Each of these three groups was then separated into four sub-groups (of 14 rats each) and after a 1 hr recuperation period (no water allowed) were injected with 9.5, 30 or 95 /zg/kg clonidine or 0.9% NaC1 SC and were allowed water and food access during the next 6 hr. Water intake was measured at the end of the period.
I1
Drug Used
i =.t
0 37.5 75 150 300 ,ug I kg CLONIDINE FIG. 1. Short-term effects of parenteral clonidine on fluid intake (Results of Experiment l). Bars refer to overall means (over 3 days and 15 rats) - SEM. Open bars: 0-24 hr, and striped bars: 0-6 hr consumption. Significance refers to unpaired t-test comparison with controls: l: 0-24 hr, 2:0-6 hr, and is denoted as: (a)p<0.001, (b)p< 0.0l, (c)p <0.05, (d) N S: p >0.05 (this notation is used throughout).
and the whole procedure was repeated (together with a measurement of body weight) 24 hr after injection, just before the next injection. The day following the 18 days of control injections was designated as the first day of the experiment proper and rats were injected each day (at 10:00) with 300 /zg/kg clonidine SC on this and the following 10 days.
Experiment 3: Influence of nephrectomy or ligation of the ureters on effects of clonidine in "thirsty" rats. Since the acute depression of water intake after clonidine, in intact rats, was followed by a bout of drinking (see below: results) the possibility that the delayed dipsogenic effect could be a consequence of the primary diuretic effect was investigated by obviating diuresis by nephrectomy. Nephrectomized animals, however, not only fail to produce urine, but also are deprived of renin, a known dipsogenic hormone. Control experiments were, therefore, carried out in rats with ligated ureters, i.e., animals unable to excrete urine but possessing a normal renal renin store. Rats of 190-210 g body weight were trained during one week to drink tap water for a period of 6 hr (10:00 to 16:00) daily. Standard rat pellets were available day and night, ad lib. Under these conditions the rats stabilized their water intake at 75% (74 _+ 2 g/kg/6 hr) of that under ad lib conditions. On the third day of stable, restricted water intake the rats were randomly distributed into
Clonidine hydrochloride was a gift of C.H. Boehringer Sohn, Ingelheim am Rhein, F.R.G. and doses are expressed as base.
Statistics Results are expressed as m e a n s - standard errors of means (SEM). Significance of differences between means was calculated by paired or unpaired t-test. Percentages are given as means +_ SEM but no t-tests were carried out on such transformed values. RESULTS
Experiment 1 The smaller doses of clonidine (37.5 and 75/zg/kg SC) had a double effect on water intake: there was an inhibition during the 6 hr following the injection (significant for 75/zg/kg) and this was followed by an enhanced rate of drinking during the next 18 hr giving an overall increase in water intake during a 24 hr period (Fig. 1). Higher doses of clonidine (150 and 300 /zg/kg SC) further diminished 6 hr water intake; " c o m p e n s a t i o n " occurred during the following 18 hr for a dose of 150/xg/kg so that the 24 hr intake did not significantly differ from that of controls; for a dose of 300/~g/kg there was less compensation and 24 hr intake was lower than that of controls. When given SC, clonidine, thus had an initial inhibitory effect on water intake (at all doses), this effect being compensated later to various degrees (dependent on dose). All doses depressed food intake (Fig. 2). A high dose (300/xg/kg) had a marked effect on both types of ingestive behavior possibly related to its sedative effect; animals lost their fighting reflexes for varying lengths of time after clonidine injections; this effect was not quantified in the present experiments.
CLONIDINE AND INGESTIVE BEHAVIOR
FOOD INTAKE gl kg law.24hs
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CLONIDINE
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75
~
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BODY WEIGHT % A 13 DAYS FIG. 2. Short-term effects of parenteral clonidine on food intake and change in body weight (Results of Experiment 1). Bars refer to overall means (for 3 days and 15 rats) _+ SEM.
Experiment 2 After each daily clonidine injection there was an increase in urine output thus, on the 1st day the 6 hr urine output was: 39.5 _ 2.0, the 24 hr urine output: 44.4 ___ 2.4 ml/kg) (controis: 5 +-- 1 and 34 ___ 6 ml/kg), but water intake did not significantly differ from control levels for the first three days: 112.5 ___ 6.3 ml/kg/24 hr (average for 3 days). On the fourth day, water intake rose to 142.9 - 19.5 ml/kg/24 hr (p<0.01 compared to control value). After the fourth day, water intake and urine volume remained elevated and the results for the final 3 days of the 10 days treatment period given in Fig. 3. During the whole treatment period, there was an increased water intake during the 6 to 24 hr following the clonidine injection, whereas a very large increase in urine flow occurred during the first 6 hr following injection. The increased water intake (26 ml/kg/24 hr) approximately compensated the initial urinary water loss (27 ml/kg/24 hr). The absolute total values of urine flow shown in Fig. 3 appear to be 2 to 4 times smaller than the rates of water intake. This apparent incongruity is due to the fact that the rates of urine flow shown were not corrected for the large evaporative loss of urine water which occurred in the collecting funnels of the metabolic cages. The evaporative loss varied inversely with the rate of urine flow over 70 to approximately 10 percent of total urine flow. Using a pump simulator of urine flow with a continuous water drop flow of 0.1 to 1.0 ml/hr, we found that the dependance of recovery of urine flow on flow.rate could be expressed as a linear regression as: (% volume recovery in 24 hr)=23 ( - 9) +60 (__ 6) ml true flow. At urine flow rates above 2 ml/hr, as observed in the first hours of diuresis after
+Oil-+ I 30
20
~
WATER FOOD URINE INTAKE INTAKE VOLUME FIG. 3. Effect of 10daystreatmentwith parenteralclonidine on food and fluid intake (Results of Experiment 2). Significance refers to paired t-test between preliminary period: injection 0.9% NaCI SC, Days -3 to -1, open columns, and test period: injection 300 tzg/kg clonidine SC, Days 8 to 10, dotted columns. First letter refers to 0--6 hr period (cross-hatched columns) and second to 0-24 hr period. parenteral clonidine, the evaporative loss of water from the urine collection funnels may be neglected. Apparent urine flow rates over 24 hr corrected by recourse to this equation yielded " t r u e " urine flow rates approximately equal to the rates of water intake over 24 hr. After 4 days of clonidine treatment there was an increase in food intake during 6 hr following each daily clonidine injection. The increase in food and fluid intake continued up to the end of the treatment period (Fig. 3). In another group of 4 rats, not described in detail here, which were given 300/zg/kg b.w. clonidine daily for a longer period, water intake (89 - 4 ml/kg b.w./day as compared to 68 _ 2 ml/kg b.w./day before treatment, and 63 _ 6 ml/kg b.w./day in saline injected controis) and urine flow (43 -+ 2 ml/kg b.w. as compared to 16 _ 1 ml/kg before treatment and 24 - 6 ml/kg b.w./day for saline injected controls) remained elevated at the end of 3 weeks of treatment.
Experiment 3 In sham-operated rats, deprived of water for 18 hr ("thirsty"), a dose of 95 /~g/kg clonidine SC depressed water-intake (Fig. 4) to the same extent as a similar dose (75 /zg/kg) in water satiated rats (Fig. 1: shaded column), while lower doses had no significant effect in either condition.
76
ATKINSON, KIRCHERTZ AND PETERS-HAEFELI d.~ , '
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FIG. 4. Influence of nephrectomy or ligation of ureters on clonidine effect on 6 hr water intake in "thirsty" rats (Results of Experiment 3). Open bars: sham-operated; Cross hatched bars: bilateral nephrectomy; Full bars: ligation of ureters; Results are given as means of 14 rats _+ SEM.
Thus in thirsty rats highly motivated to drink, clonidine inhibited thirst to the same degree as in satiated rats. The acute antidipsic effect of clonidine in intact rats presumably was partially masked by the dipsogenic effect of water loss induced by the diuretic effect of the drug: bilateral nephrectomy or ligation of both ureters, which as such, had no significant influence on the 6 hr water intake of thirsty rats (Fig. 4) enhanced the antidipsic effect of clonidine which depressed water intake at the doses of 9.5 p,g or 30/xg/kg (Fig. 4). There was no difference between bilateral nephrectomy and ligation of both ureters in this respect. Clonidine had no significant effect on food intake in the rats which were thirsty, but not hungry. Nephrectomy or ligation of ureters induced a 30-50% decrease in 6 hr food intake, irrespective of the injection received. Thus, in rats trained to drink 6 hr per day, and incapable of showing a diuretic response, clonidine is seen to have a primarily inhibitory effect on water intake. Experiment 4
In parallel to Experiment 3 described above, in this experiment rats were trained to eat 6 hr per day and were thus hungry at the moment of the clonidine or control injections. 75/~g/kg clonidine had no effect on food consumption during the 6 hr following the injection (during which period the rats had been trained to eat): controls: 63 +- 3, clonidine 57 -+ 2 g/kg; whereas 300/~g/kg clonidine decreased intake to 24 _+ 3 g/kg (p<0.001, n = 7 for all groups). Only a large dose of clonidine had an inhibitory effect on food intake in starved rats, while a smaller dose, which did depress eating in rats less motivated to do so (Experiment 2), proved ineffective in hungry animals. DISCUSSION
Effects o f Clonidine on Food Intake
In the majority of the experiments reported here,
clonidine given subcutaneously either decreased, or had no effect on, food intake. A depression of food intake by peripheral clonidine has previously been observed by Le Douarec et al. [ 11]. Only in rats previously treated with large doses of clonidine for more than 3 days, 300/xg/kg clonidine SC increased food intake during the 6 hr following its administration. Depression of food intake after peripheral clonidine contrasts with the effect of the same drug injected intracranially. When clonidine is given intracranially, it stimulates alphaadrenergic receptors in the hypothalamus and so induces feeding. Broekkamp and van Rossum [3] showed that clonidine injected into the antero-lateral hypothalamus in rats (fasted for 24 hr) in doses of 3 to 167 ixg induced a (dose-independent) increase of food intake of the same order as that induced by 23 txg/kg norepinephrine in the first hr following the injection. Phentolarnine blocked both responses. CIonidine injected into the lateral ventricle at doses of 0.13 to 8 /zg/kg increased the consumption of wet (sweetened milk) or dry food in satiated starved or amphetamine pre-treated rats [13]. Again there was no clear cut dose-response and the response was complete 30 min after the injection. Clonidine was 100 times more potent than norepinephrine. Since clonidine readily passes the blood-brain-barrier [18], peripheral clonidine should similarly enhance rather than depress eating, as actually observed. In order to explain the contradictory effects of peripheral and of intracranial clonidine on eating, Le Douarec et al. [ i l l suggested the existence of two noradrenergic receptor systems, one stimulatory and the other inhibitory. The doses of clonidine used here on the other hand, had a marked sedative effect, as previously described by other investigators [9,17] and also found by observing the animals in the present experiments. Another alpha-sympathomimetic, norepinephrine, when given intraventricularly, or systemically in animals with an immature blood brain-barrier, also induces sleep [4]. While it is evident that sleeping animals will not usually interrupt their sleep to eat, it must be realized that sedative agents usually enhance rather than depress eating [2,8]. The acute anorectic effect of clonidine also differed from that of appetite-suppressing drugs known to act on cerebral receptors, such as amphetamines, by the fact that it appeared to be depressed (Experiment 4), rather than enhanced in starved animals. It, therefore, appears more likely that clonidine, when given peripherally, but not when injected into the brain, induces signals which interfere with its own stimulating effect on food ingestion by action on hypothalamic receptors. The most likely peripheral satiety signal which clonidine could induce would be hyperglycemia due to hepatic glycogenolysis [5]. Hyperglycemia induced by glucagon, epinephrine or glucose is known to interfere with eating in response to various stimuli [15]. In only one situation, i.e., in rats pretreated with clonidine for more than 3 days, the drug actually induced a marked eating response (Fig. 3). This observation suggests that tolerance to the satiety signal generating effect of peripheral clonidine may develop more rapidly than tolerance to its cerebral orexigenic effect. Tolerance has been shown to develop against peripheral alpha-adrenergic [19], as well as against behavioral and blood-pressure lowering effects [9] of clonidine. On the other hand, it is well-known that tolerance to different effects of one drug often develops after widely different times [6].
C L O N I D I N E AND INGESTIVE BEHAVIOR
77
Effects on Drinking
When clonidine is given subcutaneously, there is an increase in 24 hr fluid intake. At low doses, (37.5 and 75/xg/kg SC in Fig. 1), this effect is seen immediately. At larger doses it occurs only after four days of clonidine treatment when tolerance may have developed to a (hypothetical) satiety signal producing effect of large doses of clonidine (300 gg/kg SC in Fig. 3). This increase in fluid intake appears to be secondary to an increase in water loss by clonidine-induced diuresis as: (a) there is substantial increase in 6 hr urine output (Fig. 3) while the increase in water intake occurs from the 6th to the 24th hr following clonidine administration; (b) in the I0 day experiment, the increase in water loss by diuresis is compensated more or less precisely, by an increase in water intake (Fig. 3) and (c) when a diuretic response to clonidine was suppressed by bilateral nephrectomy or ligation of both ureters, clonidine caused only a decrease with no delayed increase of water intake. The observations made in thirsty rats trained to drink 6 hr a day, suggest that the primary influence of clonidine on water intake is inhibitory, presumably by a stimulation of central alpha-adrenergic receptors which is known to diminish thirst [4]. In rats, made anuric either by nephrectomy or by ligation of the ureters, doses of clonidine which had no significant effect on water intake of sham-operated controls depressed water intake. It appears likely that in intact
(sham-operated) thirsty rats, the thirst, due to a primary diuretic effect overcame the primary inhibitory effect on water intake of low clonidine doses. Since bilateral nephrectomy and ligation of the ureters sensitized rats to the antidipsic effect of clonidine to the same extent, the sensitization appeared to be related to the loss of the urine excretory function of the kidneys rather than to an elimination of the dipsogenic renin-angiotensin system. This system is eliminated by bilateral nephrectomy, but not by ligating the ureters. Thirsty, anuric rats injected with 0.9% NaCl, drank as much as thirsty, sham-operated controls, thus nephrectomy or ligation of ureters alone did not diminish water intake. In conclusion, it can be stated that, clonidine, when given peripherally is: (1) a "secondary" dipsogen owing to its primary diuretic effect; (2) a primary anti-dipsogenic agent, presumably by its action on cerebral alpha-adrenergic receptors; (3) an indirect, "anorexic" agent, presumably by activation of peripheral satiety signals and; (4) an orexigenic agent in rats treated with large doses of clonidine for more than 4 days.
ACKNOWLEDGEMENTS We would like to thank Prof. G. Peters for his valuable guidance and Ms. R. Pera and P. Liithi for their excellent technical assistance.
REFERENCES
1. Atkinson, J., E. J. Kirchertz and L. Peters-Haefeli. Effects of clonidine on water intake in rats. VIth Int. Conf. Phys. Food and Fluid Intake, Pads, 1977. 2. Bignami, G., L. de Acetis and G. L. Gatti. Facilitation and impairment of avoidance responding by phenobarbital sodium, chlordiazepoxide and diazepam. J. Pharmac. exp. Ther. 176: 725-732, 1971. 3. Broekkamp, C. and J. M. van Rossum. Clonidine induced intrahypothalamic stimulation of eating in rats. Psychopharmacologia 25: 162-168, 1972. 4. Grossman S. P. Essentials of Physiological Psychology. New York: Wiley, 1973. 5. Hoefke, W. and W. Kobinger. Pharmakologische Wirkungen des 2-(2,6-Dichlorphenylamino)-2-imidazolin-hydrochlorids, einer neuen, antihypertensiven Substanz. ArzneimitteI-Forseh. 8: 1038-1050, 1966. 6. Isbell, H. and W. M. White. Clinical characteristics of addictions. Am. J. Med. 14: 558-565, 1953. 7. Kirchertz, E. J., J. Atkinson and L. Peters-Haefeli. Clonidine induced diuresis and increase of water intake in rats. Experientia 33: 807, 1977. 8. Kulkovsky, P. J., D. Porte Jr. and S. C. Woods. Elevation of rat plasma insulin by intrathecal pentobarbital. Experientia 31: 123-124, 1975. 9. Laverty, R. and K. M. Taylor. Behavioural and biochemical effects of 2-(2,6-dichlorphenylamino)-2-imidazoline hydrochloride (St 155) on the central nervous system. Br. J. Pharmac. 35: 253-264, 1969. 10. Le Douarec, J. C., H. Schmitt and B. Lucet. Influence de la clonidine et des substances a-sympathomim6tiques sur la prise d'eau chez le rat assoiff6. J. Pharmac. (Paris) 2:435 A.4~,1971.
11. Le Douarec, J. C., H. Schmitt and B, Lucet. Effets de la clonidine et d'autres agents sympathomim6tiques sur la prise de nourriture. Antagonisme par des agents adr6nolytiques. J. Pharmac. (Paris) 3: 187-198, 1972. 12. Leibowitz, S. Reciprocal "hunger"-regulatingcircuits involving c~-and B-receptors located eespectively in the ventromedial and lateral hypothalamus. Proc. natn. Acad. Sci. USA 67: 10631070, t970. 13. Ritter, S., D. Wise and L. Stein. Neurochemical regulation of feeding in rat: facilitation by a-noradrenergic, but not dopaminergic, receptor stimulants. J. comp. physiol. Psychol. 88: 778-784, 1975. 14. Rowland, N. and C. Flamm. Quinine drinking: more regulatory puzzles. Physiol. Behav. 18: 1165-1170, 1977. 15. Russek, M., A. M. Rodriguez-Zendejas and S. Pina. Hypothetical liver receptors and the anorexia caused by adrenalin and glucose. Physiol. Behav. 3: 249-257, 1968. 16. Setler, P. E. Cerebral cholinergic and adrenergic mechanisms of thirst. In: Rein et Foie 16 B (Maladies de la Nutrition), Joarnges lnternationales de N@hrologie, Vittel 1974, pp. 175-187. 17. Tilson, H. A., J. H. Chamberlain, J. A. Gylys and J. P. Buyniski. Behavioural suppressant effects of clonidine in strains of normotensive and hypertensive rats. Eur. J. Pharmac. 43: 99-105, 1977. 18. Van Zwieten, P. A. Antihypertensive drugs with a central action. Progr. Pharmac. 1: 1-63, 1975. 19. Walland, A. and W. Kobinger. On the problem of tolerance of 2-(2,6-dichlorphenylamino)-2-imidazoline. ArzneimitteI-Forsch. 21: 61-65, 1971.
Effect of Peripheral Clonidine on Ingestive Behavior JEFFREY
ATKINSON,
E R N S T J. K I R C H E R T Z
AND LISE PETERS-HAEFELI
3
Institut de Pharmacologie de l'Universit~, 1011 Lausanne, Switzerland ( R e c e i v e d 14 D e c e m b e r 1977) ATKINSON, J., E. J. KIRCHERTZ AND L. PETERS-HAEFELI. Effect of peripheral clonidine on ingestive behavior. PHYSIOL. BEHAV. 21(1) 73--77, 1978.--The effect of parenteral elonidine, a central alpha-sympathomimetic agent, on eating and drinking was studied in rats. Small (37.5-75 /~g/kg) and large (150--300/~g/kg) doses of clonidine acutely depressed water intake for periods up to 6 hours. The antidispsogenic effect was followed by a delayed increase of fluid intake apparently due to the diuretic effect of the drug in rats, and this was suppressed by nephrectomy. With dally injections for periods up to 3 days large doses depressed both water and food intake. When dally injections were continued for more than 4 days, an appetite-stimulating effect was noted. It was concluded that clonidine primarily depresses water and food intake. The delayed, apparently dipsogenic, effect observed is due to a primary diuretic effect. The appetitestimulating effect of clonidine observed with chronic treatment may be a primary cerebral effect unmasked when tolerance develops to the (presumably) peripheral, anorectic effect. Clonidine
Food intake
Fluid intake
Diuresis
IT IS known that stimulation of central alpha-adrenergic receptors will provoke eating [12] and inhibit drinking [16]. Clonidine, an imidazoline derivative used as an antihypertensive drug, should share these properties, as: (a) it acts as an alpha-adrenergic agonist [5], and (b) has a marked central action [18]. Published reports on clonidine effects on food and fluid intake are, however, contradictory. It has been reported that clonidine, given intracerebrally, stimulates [3,13] or, given peripherally, inhibits food intake [11]; likewise for fluid intake, clonidine has been reported to have no effect when given intracerebrally [13] or to decrease intake when given peripherally [10]. When attempting to elucidate these contradictions, general points such as differences in administration route, doses used, etc. have to be taken into account, together with two specific points, peculiar to the effects of clonidine. The fu-st is that s.c. clonidine has a marked sedative effect [9] and thus, will induce a dose-dependent decrease in overall activity, possibly, including food intake. The second point is that clonidine has a marked, dosedependent diuretic effect, in the rat [5], and could, therefore, be expected, to induce various degrees of secondary drinking following a primary, dose-dependent, diuretic water loss. We have attempted to study the basic actions of clonidine on food and fluid intake and their possible perturbation by sedative and/or diuretic " s i d e " effects, in the following ways: 1. Using a wide range of clonidine doses from those possessing little sedative action to those inducing complete, long-lasting immobility. 2. Using rats incapable of losing water by diuresis (bilat-
eral nephrectomy or bilateral ureter ligation). 3. Following changes in water loss and water intake when clonidine is given chronically, as it has been reported that tolerance can develop to some of its effects e.g. its peripheral alpha-sympathomimetic [ 19] and central sedative [9] actions. METHOD
Animals Female Sprague-Dawley rats were maintained on a standard rat diet and tap water ad lib before each experiment.
Procedure Experiment 1: Short-term effects of parenteral clonidine on food andfluid intake. Rats of 250--260 g body weight were injected SC once daily (at 10:00 a.m.) with doses of 37.5, 75, 150 or 300/zg/kg clonidine for 3 days. They were allowed free access to a standard rat diet (pellets) and water. F o o d intake and body weight change over 3 days, and water intake during the 6 and 24 hr following each daily injection, were measured. Controls wre injected with the same volumes of solvent (0.9% NaC1, 5 ml/kg).
Experiment 2: Effect of lO days treatment with parenteral clonidine on food andfluid intake. Eight rats o f 188 --- 2 g initial body weight were housed in siliconized, polyethylene metabolic cages. Standard rat pellets and water were continuously available ad lib. and rats were injected daily (at 10:00 a.m.) with 0.9% NaCI (5 ml/kg) SC for a preliminary period of 18 days. F o o d and water intake and urine volume were measured 6 hr later, the rats were placed in clean cages,
tPreliminary communications see [1] and [7]. 2Research supported by Swiss National Science Foundation, Grants No. 3.053-074 and 3.028-0.76. 3Requests for reprints to J. Atkinson, Institut de Pharmacologie de l'Universit~, 1011 Lausanne, Switzerland.
C o p y r i g h t © 1978 B r a i n R e s e a r c h P u b l i c a t i o n s Inc.--0031-9384/78/0701-0073502.00/0
74
A T K I N S O N , K I R C H E R T Z AND P E T E R S - H A E F E L 1
WATER
PROBABILITY c d m[Ikg b.w (~)b 130(~ d b b INTAKE
120. ll0100-
~ ] HOURS 7 TO
4
HOURS 1 TO 6 AFTER CLONIDINE S.C.
90. 80.
60. 50.
40. 30. 20.
0
Experiment 4: Short-term effects of clonidine on food intake of hungry rats. Acute anorectic effects of drugs are magnified in animals given access to food after period of starvation. Experiments were, therefore, carried out in rats routinely starved for 18/24 hr. Rats of 190 to 200 g body weight were trained to eat a standard rat diet 6 hr per day (10:00 to 16:00) with tap water for 24/24 hr, ad lib. After 3 days, they stablized their food intake at 53 --+ 2 g/kg/6 hr (water intake was 103 _ _8 g/kg/24 hr). Food intake was, thus, approximately 75% of the unrestricted 24 hr intake. On the 4th day, they were distributed at random into 3 groups (of 6 rats each) and the groups were injected with 75 or 300 /xg/kg clonidine or 0.9% NaC1 SC. Free access to food and water was allowed from the time of the injection onwards, and food intake was measured 6 hr later.
70.
10
three groups and were subjected to one of the following operations (under ether anesthesia): bilateral nephrectomy: both kidneys removed via a single dorsal incision; ligation of ureters: both ureters tied off via a single ventral incision; sham: ventral incision, separation of ureters and kidneys from other viscera. Each of these three groups was then separated into four sub-groups (of 14 rats each) and after a 1 hr recuperation period (no water allowed) were injected with 9.5, 30 or 95 /zg/kg clonidine or 0.9% NaC1 SC and were allowed water and food access during the next 6 hr. Water intake was measured at the end of the period.
I1
Drug Used
i =.t
0 37.5 75 150 300 ,ug I kg CLONIDINE FIG. 1. Short-term effects of parenteral clonidine on fluid intake (Results of Experiment l). Bars refer to overall means (over 3 days and 15 rats) - SEM. Open bars: 0-24 hr, and striped bars: 0-6 hr consumption. Significance refers to unpaired t-test comparison with controls: l: 0-24 hr, 2:0-6 hr, and is denoted as: (a)p<0.001, (b)p< 0.0l, (c)p <0.05, (d) N S: p >0.05 (this notation is used throughout).
and the whole procedure was repeated (together with a measurement of body weight) 24 hr after injection, just before the next injection. The day following the 18 days of control injections was designated as the first day of the experiment proper and rats were injected each day (at 10:00) with 300 /zg/kg clonidine SC on this and the following 10 days.
Experiment 3: Influence of nephrectomy or ligation of the ureters on effects of clonidine in "thirsty" rats. Since the acute depression of water intake after clonidine, in intact rats, was followed by a bout of drinking (see below: results) the possibility that the delayed dipsogenic effect could be a consequence of the primary diuretic effect was investigated by obviating diuresis by nephrectomy. Nephrectomized animals, however, not only fail to produce urine, but also are deprived of renin, a known dipsogenic hormone. Control experiments were, therefore, carried out in rats with ligated ureters, i.e., animals unable to excrete urine but possessing a normal renal renin store. Rats of 190-210 g body weight were trained during one week to drink tap water for a period of 6 hr (10:00 to 16:00) daily. Standard rat pellets were available day and night, ad lib. Under these conditions the rats stabilized their water intake at 75% (74 _+ 2 g/kg/6 hr) of that under ad lib conditions. On the third day of stable, restricted water intake the rats were randomly distributed into
Clonidine hydrochloride was a gift of C.H. Boehringer Sohn, Ingelheim am Rhein, F.R.G. and doses are expressed as base.
Statistics Results are expressed as m e a n s - standard errors of means (SEM). Significance of differences between means was calculated by paired or unpaired t-test. Percentages are given as means +_ SEM but no t-tests were carried out on such transformed values. RESULTS
Experiment 1 The smaller doses of clonidine (37.5 and 75/zg/kg SC) had a double effect on water intake: there was an inhibition during the 6 hr following the injection (significant for 75/zg/kg) and this was followed by an enhanced rate of drinking during the next 18 hr giving an overall increase in water intake during a 24 hr period (Fig. 1). Higher doses of clonidine (150 and 300 /zg/kg SC) further diminished 6 hr water intake; " c o m p e n s a t i o n " occurred during the following 18 hr for a dose of 150/xg/kg so that the 24 hr intake did not significantly differ from that of controls; for a dose of 300/~g/kg there was less compensation and 24 hr intake was lower than that of controls. When given SC, clonidine, thus had an initial inhibitory effect on water intake (at all doses), this effect being compensated later to various degrees (dependent on dose). All doses depressed food intake (Fig. 2). A high dose (300/xg/kg) had a marked effect on both types of ingestive behavior possibly related to its sedative effect; animals lost their fighting reflexes for varying lengths of time after clonidine injections; this effect was not quantified in the present experiments.
CLONIDINE AND INGESTIVE BEHAVIOR
FOOD INTAKE gl kg law.24hs
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70.
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--
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BODY WEIGHT % A 13 DAYS FIG. 2. Short-term effects of parenteral clonidine on food intake and change in body weight (Results of Experiment 1). Bars refer to overall means (for 3 days and 15 rats) _+ SEM.
Experiment 2 After each daily clonidine injection there was an increase in urine output thus, on the 1st day the 6 hr urine output was: 39.5 _ 2.0, the 24 hr urine output: 44.4 ___ 2.4 ml/kg) (controis: 5 +-- 1 and 34 ___ 6 ml/kg), but water intake did not significantly differ from control levels for the first three days: 112.5 ___ 6.3 ml/kg/24 hr (average for 3 days). On the fourth day, water intake rose to 142.9 - 19.5 ml/kg/24 hr (p<0.01 compared to control value). After the fourth day, water intake and urine volume remained elevated and the results for the final 3 days of the 10 days treatment period given in Fig. 3. During the whole treatment period, there was an increased water intake during the 6 to 24 hr following the clonidine injection, whereas a very large increase in urine flow occurred during the first 6 hr following injection. The increased water intake (26 ml/kg/24 hr) approximately compensated the initial urinary water loss (27 ml/kg/24 hr). The absolute total values of urine flow shown in Fig. 3 appear to be 2 to 4 times smaller than the rates of water intake. This apparent incongruity is due to the fact that the rates of urine flow shown were not corrected for the large evaporative loss of urine water which occurred in the collecting funnels of the metabolic cages. The evaporative loss varied inversely with the rate of urine flow over 70 to approximately 10 percent of total urine flow. Using a pump simulator of urine flow with a continuous water drop flow of 0.1 to 1.0 ml/hr, we found that the dependance of recovery of urine flow on flow.rate could be expressed as a linear regression as: (% volume recovery in 24 hr)=23 ( - 9) +60 (__ 6) ml true flow. At urine flow rates above 2 ml/hr, as observed in the first hours of diuresis after
+Oil-+ I 30
20
~
WATER FOOD URINE INTAKE INTAKE VOLUME FIG. 3. Effect of 10daystreatmentwith parenteralclonidine on food and fluid intake (Results of Experiment 2). Significance refers to paired t-test between preliminary period: injection 0.9% NaCI SC, Days -3 to -1, open columns, and test period: injection 300 tzg/kg clonidine SC, Days 8 to 10, dotted columns. First letter refers to 0--6 hr period (cross-hatched columns) and second to 0-24 hr period. parenteral clonidine, the evaporative loss of water from the urine collection funnels may be neglected. Apparent urine flow rates over 24 hr corrected by recourse to this equation yielded " t r u e " urine flow rates approximately equal to the rates of water intake over 24 hr. After 4 days of clonidine treatment there was an increase in food intake during 6 hr following each daily clonidine injection. The increase in food and fluid intake continued up to the end of the treatment period (Fig. 3). In another group of 4 rats, not described in detail here, which were given 300/zg/kg b.w. clonidine daily for a longer period, water intake (89 - 4 ml/kg b.w./day as compared to 68 _ 2 ml/kg b.w./day before treatment, and 63 _ 6 ml/kg b.w./day in saline injected controis) and urine flow (43 -+ 2 ml/kg b.w. as compared to 16 _ 1 ml/kg before treatment and 24 - 6 ml/kg b.w./day for saline injected controls) remained elevated at the end of 3 weeks of treatment.
Experiment 3 In sham-operated rats, deprived of water for 18 hr ("thirsty"), a dose of 95 /~g/kg clonidine SC depressed water-intake (Fig. 4) to the same extent as a similar dose (75 /zg/kg) in water satiated rats (Fig. 1: shaded column), while lower doses had no significant effect in either condition.
76
ATKINSON, KIRCHERTZ AND PETERS-HAEFELI d.~ , '
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FIG. 4. Influence of nephrectomy or ligation of ureters on clonidine effect on 6 hr water intake in "thirsty" rats (Results of Experiment 3). Open bars: sham-operated; Cross hatched bars: bilateral nephrectomy; Full bars: ligation of ureters; Results are given as means of 14 rats _+ SEM.
Thus in thirsty rats highly motivated to drink, clonidine inhibited thirst to the same degree as in satiated rats. The acute antidipsic effect of clonidine in intact rats presumably was partially masked by the dipsogenic effect of water loss induced by the diuretic effect of the drug: bilateral nephrectomy or ligation of both ureters, which as such, had no significant influence on the 6 hr water intake of thirsty rats (Fig. 4) enhanced the antidipsic effect of clonidine which depressed water intake at the doses of 9.5 p,g or 30/xg/kg (Fig. 4). There was no difference between bilateral nephrectomy and ligation of both ureters in this respect. Clonidine had no significant effect on food intake in the rats which were thirsty, but not hungry. Nephrectomy or ligation of ureters induced a 30-50% decrease in 6 hr food intake, irrespective of the injection received. Thus, in rats trained to drink 6 hr per day, and incapable of showing a diuretic response, clonidine is seen to have a primarily inhibitory effect on water intake. Experiment 4
In parallel to Experiment 3 described above, in this experiment rats were trained to eat 6 hr per day and were thus hungry at the moment of the clonidine or control injections. 75/~g/kg clonidine had no effect on food consumption during the 6 hr following the injection (during which period the rats had been trained to eat): controls: 63 +- 3, clonidine 57 -+ 2 g/kg; whereas 300/~g/kg clonidine decreased intake to 24 _+ 3 g/kg (p<0.001, n = 7 for all groups). Only a large dose of clonidine had an inhibitory effect on food intake in starved rats, while a smaller dose, which did depress eating in rats less motivated to do so (Experiment 2), proved ineffective in hungry animals. DISCUSSION
Effects o f Clonidine on Food Intake
In the majority of the experiments reported here,
clonidine given subcutaneously either decreased, or had no effect on, food intake. A depression of food intake by peripheral clonidine has previously been observed by Le Douarec et al. [ 11]. Only in rats previously treated with large doses of clonidine for more than 3 days, 300/xg/kg clonidine SC increased food intake during the 6 hr following its administration. Depression of food intake after peripheral clonidine contrasts with the effect of the same drug injected intracranially. When clonidine is given intracranially, it stimulates alphaadrenergic receptors in the hypothalamus and so induces feeding. Broekkamp and van Rossum [3] showed that clonidine injected into the antero-lateral hypothalamus in rats (fasted for 24 hr) in doses of 3 to 167 ixg induced a (dose-independent) increase of food intake of the same order as that induced by 23 txg/kg norepinephrine in the first hr following the injection. Phentolarnine blocked both responses. CIonidine injected into the lateral ventricle at doses of 0.13 to 8 /zg/kg increased the consumption of wet (sweetened milk) or dry food in satiated starved or amphetamine pre-treated rats [13]. Again there was no clear cut dose-response and the response was complete 30 min after the injection. Clonidine was 100 times more potent than norepinephrine. Since clonidine readily passes the blood-brain-barrier [18], peripheral clonidine should similarly enhance rather than depress eating, as actually observed. In order to explain the contradictory effects of peripheral and of intracranial clonidine on eating, Le Douarec et al. [ i l l suggested the existence of two noradrenergic receptor systems, one stimulatory and the other inhibitory. The doses of clonidine used here on the other hand, had a marked sedative effect, as previously described by other investigators [9,17] and also found by observing the animals in the present experiments. Another alpha-sympathomimetic, norepinephrine, when given intraventricularly, or systemically in animals with an immature blood brain-barrier, also induces sleep [4]. While it is evident that sleeping animals will not usually interrupt their sleep to eat, it must be realized that sedative agents usually enhance rather than depress eating [2,8]. The acute anorectic effect of clonidine also differed from that of appetite-suppressing drugs known to act on cerebral receptors, such as amphetamines, by the fact that it appeared to be depressed (Experiment 4), rather than enhanced in starved animals. It, therefore, appears more likely that clonidine, when given peripherally, but not when injected into the brain, induces signals which interfere with its own stimulating effect on food ingestion by action on hypothalamic receptors. The most likely peripheral satiety signal which clonidine could induce would be hyperglycemia due to hepatic glycogenolysis [5]. Hyperglycemia induced by glucagon, epinephrine or glucose is known to interfere with eating in response to various stimuli [15]. In only one situation, i.e., in rats pretreated with clonidine for more than 3 days, the drug actually induced a marked eating response (Fig. 3). This observation suggests that tolerance to the satiety signal generating effect of peripheral clonidine may develop more rapidly than tolerance to its cerebral orexigenic effect. Tolerance has been shown to develop against peripheral alpha-adrenergic [19], as well as against behavioral and blood-pressure lowering effects [9] of clonidine. On the other hand, it is well-known that tolerance to different effects of one drug often develops after widely different times [6].
C L O N I D I N E AND INGESTIVE BEHAVIOR
77
Effects on Drinking
When clonidine is given subcutaneously, there is an increase in 24 hr fluid intake. At low doses, (37.5 and 75/xg/kg SC in Fig. 1), this effect is seen immediately. At larger doses it occurs only after four days of clonidine treatment when tolerance may have developed to a (hypothetical) satiety signal producing effect of large doses of clonidine (300 gg/kg SC in Fig. 3). This increase in fluid intake appears to be secondary to an increase in water loss by clonidine-induced diuresis as: (a) there is substantial increase in 6 hr urine output (Fig. 3) while the increase in water intake occurs from the 6th to the 24th hr following clonidine administration; (b) in the I0 day experiment, the increase in water loss by diuresis is compensated more or less precisely, by an increase in water intake (Fig. 3) and (c) when a diuretic response to clonidine was suppressed by bilateral nephrectomy or ligation of both ureters, clonidine caused only a decrease with no delayed increase of water intake. The observations made in thirsty rats trained to drink 6 hr a day, suggest that the primary influence of clonidine on water intake is inhibitory, presumably by a stimulation of central alpha-adrenergic receptors which is known to diminish thirst [4]. In rats, made anuric either by nephrectomy or by ligation of the ureters, doses of clonidine which had no significant effect on water intake of sham-operated controls depressed water intake. It appears likely that in intact
(sham-operated) thirsty rats, the thirst, due to a primary diuretic effect overcame the primary inhibitory effect on water intake of low clonidine doses. Since bilateral nephrectomy and ligation of the ureters sensitized rats to the antidipsic effect of clonidine to the same extent, the sensitization appeared to be related to the loss of the urine excretory function of the kidneys rather than to an elimination of the dipsogenic renin-angiotensin system. This system is eliminated by bilateral nephrectomy, but not by ligating the ureters. Thirsty, anuric rats injected with 0.9% NaCl, drank as much as thirsty, sham-operated controls, thus nephrectomy or ligation of ureters alone did not diminish water intake. In conclusion, it can be stated that, clonidine, when given peripherally is: (1) a "secondary" dipsogen owing to its primary diuretic effect; (2) a primary anti-dipsogenic agent, presumably by its action on cerebral alpha-adrenergic receptors; (3) an indirect, "anorexic" agent, presumably by activation of peripheral satiety signals and; (4) an orexigenic agent in rats treated with large doses of clonidine for more than 4 days.
ACKNOWLEDGEMENTS We would like to thank Prof. G. Peters for his valuable guidance and Ms. R. Pera and P. Liithi for their excellent technical assistance.
REFERENCES
1. Atkinson, J., E. J. Kirchertz and L. Peters-Haefeli. Effects of clonidine on water intake in rats. VIth Int. Conf. Phys. Food and Fluid Intake, Pads, 1977. 2. Bignami, G., L. de Acetis and G. L. Gatti. Facilitation and impairment of avoidance responding by phenobarbital sodium, chlordiazepoxide and diazepam. J. Pharmac. exp. Ther. 176: 725-732, 1971. 3. Broekkamp, C. and J. M. van Rossum. Clonidine induced intrahypothalamic stimulation of eating in rats. Psychopharmacologia 25: 162-168, 1972. 4. Grossman S. P. Essentials of Physiological Psychology. New York: Wiley, 1973. 5. Hoefke, W. and W. Kobinger. Pharmakologische Wirkungen des 2-(2,6-Dichlorphenylamino)-2-imidazolin-hydrochlorids, einer neuen, antihypertensiven Substanz. ArzneimitteI-Forseh. 8: 1038-1050, 1966. 6. Isbell, H. and W. M. White. Clinical characteristics of addictions. Am. J. Med. 14: 558-565, 1953. 7. Kirchertz, E. J., J. Atkinson and L. Peters-Haefeli. Clonidine induced diuresis and increase of water intake in rats. Experientia 33: 807, 1977. 8. Kulkovsky, P. J., D. Porte Jr. and S. C. Woods. Elevation of rat plasma insulin by intrathecal pentobarbital. Experientia 31: 123-124, 1975. 9. Laverty, R. and K. M. Taylor. Behavioural and biochemical effects of 2-(2,6-dichlorphenylamino)-2-imidazoline hydrochloride (St 155) on the central nervous system. Br. J. Pharmac. 35: 253-264, 1969. 10. Le Douarec, J. C., H. Schmitt and B. Lucet. Influence de la clonidine et des substances a-sympathomim6tiques sur la prise d'eau chez le rat assoiff6. J. Pharmac. (Paris) 2:435 A.4~,1971.
11. Le Douarec, J. C., H. Schmitt and B, Lucet. Effets de la clonidine et d'autres agents sympathomim6tiques sur la prise de nourriture. Antagonisme par des agents adr6nolytiques. J. Pharmac. (Paris) 3: 187-198, 1972. 12. Leibowitz, S. Reciprocal "hunger"-regulatingcircuits involving c~-and B-receptors located eespectively in the ventromedial and lateral hypothalamus. Proc. natn. Acad. Sci. USA 67: 10631070, t970. 13. Ritter, S., D. Wise and L. Stein. Neurochemical regulation of feeding in rat: facilitation by a-noradrenergic, but not dopaminergic, receptor stimulants. J. comp. physiol. Psychol. 88: 778-784, 1975. 14. Rowland, N. and C. Flamm. Quinine drinking: more regulatory puzzles. Physiol. Behav. 18: 1165-1170, 1977. 15. Russek, M., A. M. Rodriguez-Zendejas and S. Pina. Hypothetical liver receptors and the anorexia caused by adrenalin and glucose. Physiol. Behav. 3: 249-257, 1968. 16. Setler, P. E. Cerebral cholinergic and adrenergic mechanisms of thirst. In: Rein et Foie 16 B (Maladies de la Nutrition), Joarnges lnternationales de N@hrologie, Vittel 1974, pp. 175-187. 17. Tilson, H. A., J. H. Chamberlain, J. A. Gylys and J. P. Buyniski. Behavioural suppressant effects of clonidine in strains of normotensive and hypertensive rats. Eur. J. Pharmac. 43: 99-105, 1977. 18. Van Zwieten, P. A. Antihypertensive drugs with a central action. Progr. Pharmac. 1: 1-63, 1975. 19. Walland, A. and W. Kobinger. On the problem of tolerance of 2-(2,6-dichlorphenylamino)-2-imidazoline. ArzneimitteI-Forsch. 21: 61-65, 1971.